Molecular Biological Studies on the Regulation of Sperm Motility

我们发现，在鲑鱼精子从精巢到精子管的运输过程中，外界pH和HCO3的升高会引起pH_i和cAMP的升高，从而影响鞭毛、轴丝的运动机构，从而获得精子运动的潜力。关于鲑鱼产卵时精子运动的启动，我们利用制备的15 kDa运动启动磷蛋白(MIPP)的单抗，首次发现鞭毛运动的控制发生在鞭毛的有限部分，即鞭毛的基底部。在淡水鲤科鱼类和海水鱼类中，我们显示了不同的精子活力启动机制：外渗透压的降低或增加导致K^+_i的增加或降低，从而导致启动轴线运动来赋予精子活力。在激活精子活力方面，用凝胶过滤和等电聚焦层析对鲱鱼卵释放的激活精子活力的物质进行了纯化，鉴定出5种蛋白质，其相对分子质量为8.1 KDa，Pl为4.9~5.4。克隆了含有Pl 5.1的HSAP的全长基因，并确定了73个氨基酸残基的氨基酸序列。HSAP类似于类胰蛋白酶抑制剂，提示类胰蛋白酶抑制因子与精子表面类胰酶受体的相互作用是触发鲱鱼精子活力激活的信号转导机制。用几种高效液相色谱分离纯化了刺参卵子释放的精子激活和吸引因子(SAAF)，认为一个分子具有激活精子和吸引精子两种功能。进一步的发现表明，精子的激活既需要钙离子和cAMP，也需要cAMP，而精子趋化只需要钙离子，这表明一个分子触发了两个不同的信号转导系统。

We found that during transit of sperm from the testis to the sperm duct of salmonid fishes, increases in external pH and HCO_3 cause increases in pH_i and cAMP which affect the motile apparatus of flagellum, axoneme, causing the acquisition of the potential for sperm motility. Concerning the initiation of sperm motility in salmonid fishes at spawning, we obtained the first finding that control of flagellar movement occurs at the limited portion i.e.basal part of flagellum using prepared monoclonal antibodies of the 15kDa motility initiating phosphoprotein (MIPP) with a molecular weight of 15KDa. In the freshwater cyprinid fishes and marine fishes, we showed the different sperm motility initiation mechanisms ; Decrease or increase in external osmolalities causes increase or decrease in K^+_i causing the initiation axonemal movement to confer motility to spermatozoa. In the activation of sperm motility, the substance released from herring egg which activate sperm motility were purified by using gel-filtration and isoelectric focusing chromatograpies and identified 5 proteins with MW of 8.1 KDa and with Pl of 4.9-5.4. cDNA of the HSAP with Pl 5.1 was cloned and amino acid sequence with 73 amino acid residues was defined. The HSAP is resemble to typsin inhibitors, suggesting that interaction between trypsin inhibitor-like HSAPs and trypsin-like receptor on the surface of spermatozoa causes the signal transduction mechanism which triggers the activation of herring sperm motility. The sperm activating and attracting factor (SAAF) released from the egg of Ciona was purified using several HPLC chromatographies and it was concluced that one molecule has two functions, sperm activation and sperm attraction. Further findings suggest that sperm activation requires both Ca^<2+> and cAMP,but sperm chemotaxis needs only Ca^<2+>, suggesting that one molecule triggers two different signal transduction systems.